POLARIZED SECRETION FROM INJURED AIRWAY EPITHELIUM

呼吸道上皮既作为极化屏障， 吸入的病原体和毒素，以及作为涉及的几种介质的来源， 对气道损伤的最初反应。 纤维连接蛋白，一种细胞外 基质蛋白被认为在气道上皮细胞中起着不可或缺的作用， 修复. 在体外，气道上皮细胞增加合成和分泌 转化生长因子-β（TGF-β）刺激后纤维连接蛋白的变化 β），一种细胞因子，被认为在组织修复中起着至关重要的作用， 调节细胞生长和分化。 候选人花费了 过去两年来， TGF-β对气管上皮细胞纤连蛋白剪接和分泌影响 细胞和拟议的实验是一个合乎逻辑的延伸， research. 首先，候选人将检查分泌物和替代品 使用技术在各种极化细胞类型中剪接纤连蛋白 例如免疫沉淀、免疫印迹和北方分析。 其次，将进行使用碘化TGF-β的交联实验 为了确定对TGF-β的极化反应是否 通过TGF-β受体的不同表面表达介导。 接下来， 该候选人建议开发一种使用纤连蛋白的转染系统， 微基因构建，以确认和进一步研究 TGF-β对纤连蛋白选择性剪接的影响。 转染 纤连蛋白表达构建体将用于确认和探索 EIIIA结构域作为靶向信号的作用。

The respiratory epithelium serves both as a polarized barrier against inhaled pathogens and toxins and as a source of several mediators involved in the initial response to airway injury. Fibronectin, an extracellular matrix protein, is believed to play an integral role in airway epithelial repair. In vitro, airway epithelial cells increase synthesis and secretion of fibronectin after stimulation with transforming growth factor-beta (TGF- beta), a cytokine believed to play a crucial role in tissue repair by modulating cellular growth and differentiation. The candidate has spent the last two years investigating the polarized regulation of alternative splicing and secretion of fibronectin by TGF-beta in tracheal epithelial cells and the proposed experiments are a logical extension of this research. First, the candidate will examine the secretion and alternative splicing of fibronectin in various polarized cell types using techniques such as immunoprecipitation, immunoblotting, and northern analysis. Second, crosslinking experiments using iodinated TGF-beta will be performed in order to determine whether the polarized response to TGF-beta is mediated by differential surface expression of TGF-beta receptors. Next, the candidate proposes to develop a transfection system using a fibronectin minigene construct in order to confirm and further study the effects of TGF-beta on alternative splicing of fibronectin. Transfection of fibronectin expression constructs will then be used to confirm and explore the role of the EIIIA domain as a targeting signal.